Compositions and methods relating to the treatment of diseases

ABSTRACT

The present invention relates to compositions and methods for promoting the induction of a cell-mediated immune response (such as that mediated by Th1 cells) and the suppression of a humoral or allergic immune response (such as that mediated by Th2 and Th17 cells). In particular, the invention relates to compositions and methods for preventing or treating allergy, such as food allergy, and associated allergic diseases, and conditions where an exaggerated Th17 response plays a detrimental role, such as inflammatory responses and autoimmune diseases. The invention further extends to the use of the compositions of the invention in the treatment and/or prophylaxis of allergy and associated allergic diseases and also of cancer.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for promotingthe induction of a cell-mediated immune response (such as that mediatedby Th1 cells) and the suppression of a humoral or allergic immuneresponse (such as that mediated by Th2 and Th17 cells). In particular,the invention relates to compositions and methods for preventing ortreating allergy, such as food allergy, and associated allergicdiseases, and conditions where an exaggerated Th17 response plays adetrimental role, such as inflammatory responses and autoimmunediseases. The invention further extends to the use of the compositionsof the invention in the treatment and/or prophylaxis of allergy andassociated allergic diseases and also of cancer.

BACKGROUND TO THE INVENTION

It is hypothesised that in certain circumstances, the Th1 response orthe Th2/Th17 response can cause disease. An over-reactive Th1 responsecan generate organ-specific autoimmune disease such as arthritis,multiple sclerosis, or Type I diabetes, while an over-reactive Th2/Th17response may underlie allergy and atrophy. It is currently believed thatTh17 cells play a major role in host defence against pathogens and anexaggerated Th17 response may lead to severe inflammatory responses andautoimmune diseases—inflammatory bowel diseases (IBD), namely,ulcerative colitis (UC) and Crohn's disease (CD), are chronicinflammatory processes of the gastrointestinal tract. In these diseasesa disturbed and exaggerated immune response, mainly towards theendogenous microflora, plays a major role. IL-17 expression is increasedin both UC and CD. Type I IFNs have been studied in clinical trials inpatients with UC and demonstrated efficacy in selected studies. Asanti-viral cytokines, it is now known that Type I IFNs can regulate thedevelopment of Th17 cells.

It is known that different pathogens induce different IFN-α subtypes invitro and that IFN-α subtypes have different antiviral,antiproliferative and immunomodulatory activities. Infection via avariety of routes, including orally, has been shown to induce differentsubtype profiles. IFN-α subtypes bind to the same receptor, activatecommon signaling pathways and are expected to have the same biologicalfunctions. All IFN-α subtypes have anti-viral activities, by definition,although their absolute efficacy in this context may vary considerably.In addition, many other biological properties have been described, butwith varying potencies, including immunomodulatory andanti-proliferative activities. The pleiotropic effects appear to be dueto differential interaction with the receptor chains and signalingthrough different intracellular pathways to an array of effectormolecules. The Type I IFN receptor consists of two chains, IFNR1 andIFNR2. There is a range of binding affinities for each of the 12 IFN-αsubtypes with the different receptor chains.

IFN-α may have a key role in the regulation of the Th1 response. It hasbeen shown that IFN-α treatment promotes Th1 cell differentiationindirectly (largely via IFN-γ), but also appears to suppress Th2 celldevelopment through the suppression of IL-4 and IL-13 gene expression.IFN-α therefore is able to re-establish a Th1/Th2 population balance indiseases and infections that promote a Th2 cell imbalance. In recentyears, it became evident that besides its anti-viral effects, severalimmunomodulatory functions are exerted by IFN-α. IFN-α can impact ondendritic cell differentiation and controls the expression of variouspro-inflammatory cytokines such as IL-8 or IL-18 and induces severalanti-inflammatory mediators such as IL-1 receptor antagonist (IL-1Ra),soluble TNF receptor p55, IL-10 and IL-18 binding protein. However, themechanisms of actions of IFN-α, and in particular individual IFN-αsubtypes, are still only partly understood.

In patients with allergy or allergic disease, a Th2-predominant immuneresponse is generated. Th2 cells secrete IL-4 and IL-13 driving B cellsto produce Immunoglobulin E (IgE) antibodies specific to an allergen. Anallergen is an antigen capable of stimulating a type-I hypersensitivityreaction in atopic individuals mainly through Immunoglobulin E(IgE)-mediated responses. Following that, IgE binds to its high affinityreceptor on mast cells, skin cells and mucosal tissues. Upon exposure tothe allergen, mast cells release their contents, which includehistamine, leukotrienes and prostaglandins. This causes allergicsymptoms including, but not limited to, red eyes, itchiness, runny nose,eczema, urticaria, angioedema, shortness of breath, wheezing, coughing,an asthma attack, abdominal pain, vomiting, diarrhoea or evenanaphylaxis.

Allergic diseases are among the most common form of chronic illness. TheWorld Health Organisation estimates that over 20 percent of the worldpopulation is affected and Europe alone has over 80 million sufferers(Global Allergy and Asthma European Network, 2008). An allergic reactionis usually caused by hypersensitivity of the immune system to anallergen, causing a misdirected immune response. Mild allergies, such ashay fever, are very common in the human population. Severe allergies canbe caused by dietary allergens, such as food, by environmentalallergens, such as the venom of stinging insects, by medication or canbe genetically determined.

Food allergy is a major health concern, which is estimated to affectaround 6% of young children and 3-4% of adults in Western societies.Food allergy is hypothesised to result from a breakdown in oraltolerance to ingested antigens or allergens. Food allergies andassociated allergic diseases include, but are not limited to, dairy(milk) allergy, including Heiner syndrome, egg allergy, soya allergy,fish (shellfish) allergy, peanut and tree nut allergy, sesame and otherseed allergy, gluten (wheat) and grains allergy, fruit and vegetableallergy, caffeine allergy, oral allergy syndrome, alcohol allergy,pollen food allergy syndrome, eosinophilic gastroenteritis, IgE mediatedgastrointestinal food allergy and C1 esterase deficiency.

Management and treatment of allergic disease is usually via threegeneral approaches: (i) avoidance of the allergen; (ii) medications thattarget disease symptoms and (iii) conventional immunotherapy, known asdesensitisation, which aims to enhance the Th1 response in establisheddisease. However, these approaches are far from ideal. Avoidance ofallergens is not always possible, medications that target diseasesymptoms, such as anti-histamines, provide only short-term relief anddesensitisation involves the use of the actual allergen, which canresult in potentially frequent harmful side-effects. The possibility ofanaphylaxis is never completely eliminated in patients suffering fromallergic diseases and this causes a great deal of stress to the patientand their families.

Interferon subtypes IFN-α10 and IFN-α14 and hybrids thereof arediscussed in PCT Publication Number WO2014/037717 and PCT ApplicationNumber PCT/GB2015/050717. In particular IFN-α10-IFN-α14 hybrids aredisclosed that contain sequences characteristic of the IFN-α10 andIFN-α14 subtype binding sites based on a consensus backbone sequence ofall 12 alpha-interferons, for exampleCDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQFQKAQAISVLHEM

QQTFNLFSTE

SSAAWEQ

LLEKFS

EL

QQ

NDLEACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLIERKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD (SEQ ID NO:3). It wouldbe advantageous to provide alternative hybrids and further compositionsand methods that provide alternative immunotherapeutic approaches.

The present inventor submits that it would be desirable to develop animproved immunotherapeutic approach which involves safer use of anallergen, as lower doses may be employed, and provides longer-termprotection against the allergic reaction. Since allergy results fromover-reactivity of Th2/Th17 cells and a corresponding lack of activityof the Th1 response, a medication that is able to modify and balance amisdirected Th2/Th17 response would be beneficial in preventing theallergic reaction. Such a medication would further be suitable to treatdiseases and conditions where an exaggerated Th17 response plays a role,such as inflammatory bowel diseases. Additionally, the inventorconsiders the ability to enhance of a Th1-mediated immune response andsuppress a Th2/Th17-mediated immune response would be useful in theprovision of compositions that mediate immune response in subjects withcancer.

SUMMARY OF THE INVENTION

The present invention relates to the action of cytokines that promotethe induction of a cell-mediated immune response (such as that mediatedby Th1 cells) and cytokines that suppress a humoral or allergic immuneresponse (such as that mediated by Th2 and Th17 cells). The presentinvention relates to hybrids of specific interferon (IFN) subtypes, andin particular to hybrids of IFN-α10 and IFN-α14.

Following extensive experimentation, the present inventor has made thesurprising discovery that the administration of a specific interferonalpha (IFN-α) subtype which is a hybrid of IFN-α10 and IFN-α14,preferably wherein the hybrid includes the primary interferon receptorbinding sites of IFN-α10 and IFN-α14 as part of a composition tomodulate the immune system, such as a vaccine, for example comprising anallergen, can result in enhanced activation of the Th1 immune responseand suppression of the Th2/Th17 immune response. In particular, theinventor has developed hybrids of IFN-α10 and IFN-α14 containing higheraffinity binding sites derived from each of IFN-α10 and IFN-α14 for theinterferon receptors IFNR1 and IFNR2. In particular, the inventordemonstrates that it is advantageous to provide IFN-α10-IFN-α14 hybridswith high affinity binding sites derived from IFN-α10 and IFN-α14subtypes that are not based on a consensus sequence of all 12 IFN-αsubtypes which provide SEQ ID NO:3. The hybrids of the present inventionare not based on a consensus backbone sequence of all 12interferon-alphas. Instead, they derive the sequence characteristics ofIFN-α10 and IFN-α14 subtypes without the sequence characteristics of theother 10 interferon-alpha subtypes.

In particular, the inventor has discovered that IFN-α10-IFN-α14 hybridsequence comprising or consisting of at least one mutation selected fromamino acids at positions 94, 101, 102, 109 or 144, (numbering as used inFIG. 16), in particular 94, 109 or 144 or combinations thereof,preferably at least two mutations selected from amino acids at positions94, 101, 102, 109 or 144, more preferably at least three mutationsselected from amino acids at positions 94, 101, 102, 109 or 144, morepreferably at least four mutations selected from amino acids atpositions 94, 101, 102, 109 or 144 or more preferably at least fivemutations selected from amino acids at positions 94, 101, 102, 109 or144 or in particular SEQ ID NO:1 result in higher affinity binding ofthe interferon receptors IFNR1 and IFNR2. In embodiments, theIFN-α10-IFN-α14 hybrid sequence can be SEQ ID NO:3 further comprisingthe mutation(s) discussed above. These hybrid sequences can be used inall aspects and embodiments of the invention.

The inventor has surprisingly discovered that administration of thenovel IFN-α10-IFN-α14 hybrids result in a greater reduction of IL-17compared to previous IFN-α10-IFN-α14 hybrids. The inventor hasdiscovered that administration of the novel IFN-α10-IFN-α14 hybridsresult in a 10%, preferably a 20%, preferably a 30%, preferably a 40%and more preferably a 50% greater reduction of IL-17 compared toprevious IFN-α10-IFN-α14 hybrids.

This has led to the identification by the inventor of improvedtherapeutic compositions which have utility in the treatment and/orprophylaxis of allergy and allergic diseases and diseases and conditionswhere an exaggerated Th17 response plays a role and also to cancer. Inparticular, the inventor has identified that the administration of atleast one food allergen which is capable of mediating a Th2/Th17 immuneresponse with a hybrid of IFN-α10 and IFN-α14 preferably wherein thehybrid includes the primary interferon receptor binding sites of IFN-α10and IFN-α14 can be used in the treatment of food allergy and associatedallergic diseases.

Moreover, the inventor has identified that the administration of atumour antigen, either a tumour associated or a tumour specific antigen,in combination with a specific interferon alpha (IFN-α) subtype which isa hybrid of IFN-α10 and IFN-α14, preferably wherein the hybrid includesthe primary interferon receptor binding sites of IFN-α10 and IFN-α14 aspart of a composition to modulate the immune system, such as a vaccine,can be used in the treatment of cancer. Suitably, the cancer may behepatic cancer, lung cancer, in particular non-small cell lung cancer,ovarian cancer, breast cancer, skin cancer, melanoma or genitourinarycancer. Suitably, the tumour associated antigen may be selected from aprostate tumour, a renal cell tumour and a bladder tumour.

Accordingly a first aspect of the present invention provides a methodfor the treatment and/or prophylaxis of a condition where an enhancementof a Th1-mediated immune response and suppression of a Th2/Th17-mediatedimmune response are desired, said method comprising the step of:

-   (i) administering to a subject in need thereof a therapeutically    effective amount of at least one interferon alpha subtype which is a    hybrid of IFN-α10 and IFN-α14 wherein the hybrid includes the    primary interferon receptor binding sites of IFN-α10 and IFN-α14.

Whilst not wishing to be bound by theory, the inventor believes thatproteins comprising the amino acid sequence of IFN-α10 have greateraffinity to interferon receptor 2 (IFNR2) and proteins comprising theamino acid sequence of IFN-α14 have greater affinity to interferonreceptor 1 (IFNR1). Thus, substitution of a protein comprising anIFN-α10 amino acid sequence with amino acids of IFN-α14 which allowbinding to interferon receptor 1 or substitution of a protein comprisingan IFN-α14 amino acid sequence with amino acids of IFN-α10 which allowbinding to interferon receptor 2 is considered to provide a IFN-α10IFN-α14 hybrid protein which should have stronger binding affinity toboth interferon receptors 1 and 2 than IFN-α10 or IFN-α14 alone. Byincluding the primary interferon receptor binding sites of IFN-α10 andIFN-α14 is meant that the hybrid comprises amino acids selected fromIFN-α10 and substituted into an IFN-α14 amino acid sequence to improvethe ability of an IFN-α14 subtype to bind to an interferon receptor 2and/or that the hybrid comprises amino acids selected from IFN-α14 andsubstituted into an IFN-α10 amino acid sequence to improve the abilityof an IFN-α10 subtype to bind to an interferon receptor 1.

Suitably, several amino acid substitutions of protein comprising anIFN-α10 amino acid sequence with amino acids of IFN-α14 determined to beinvolved in binding to interferon receptor 1 may enhance the binding ofthe protein to interferon receptor 1. Suitably, an amino acidsubstitution of protein comprising an IFN-α14 amino acid sequence withamino acids of IFN-α10 determined to be involved in binding tointerferon receptor 2 may enhance the binding of the protein tointerferon receptor 2.

In embodiments the IFN-α10-IFN-α14 hybrid can substantially have theamino-acid sequence of IFN-α10, but be modified in a region betweenamino residues 80 to 150, or suitably between amino acid residues 84 to144, or suitably amino acid residues 92 to 115 or suitably between aminoacid residues 90 to 110, (utilizing the numbering of the IFN-α10sequence providing in FIG. 16) to provide the amino acids provided bythe IFN-α14 sequence. It is considered the amino acid residues in theseregions or parts of these regions provide for the binding of IFN-α14 tointerferon receptor 1. In particular, the hybrid sequence may include atleast one, at least two, at least three, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10 or at least 11modifications of the IFN-α10 sequence to provide the correspondingresidues of the IFN-α14 sequence (suitably substituted residues arenoted in bold in FIG. 9) or a conserved mutation thereof. Inembodiments, eleven modifications are provided as indicated by the aminoacids noted in bold in FIG. 9. In embodiments, the IFN-α10-IFN-α14hybrid sequence may include at least one mutation selected from aminoacids at positions 94, 101, 102, 109 or 144, preferably at least twomutations selected from amino acids at positions 94, 101, 102, 109 or144, more preferably at least three mutations selected from amino acidsat positions 94, 101, 102, 109 or 144, more preferably at least fourmutations selected from amino acids at positions 94, 101, 102, 109 or144 or more preferably at least five mutations selected from amino acidsat positions 94, 101, 102, 109 or 144. In alternative embodiments,IFN-α14 can be utilised as a backbone structure of the hybrid and theresidues which differ between the IFN-α10 and IFN-α14 sequences at the Nand C terminal regions of the sequences can be provided in the hybridsequence as those present in the IFN-α10 sequence. Suitably at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10 or at least 11 substitutions of theIFN-α14 N-terminal sequence may be made to provide the hybrid sequenceto provide residues from IFN-α10 at those amino acid positions whereinthe amino acids are not shared/common between IFN-α10 and IFN-α14.Suitably, at least 1, at least 2, or 3 substitutions are provided at theIFN-α14 C terminal sequence to provide residues from IFN-α10 to thehybrid sequence at those amino acid positions which are notshared/common between IFN-α10 and IFN-α14. In embodiments at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10 or at least 11 substitutions from theN-terminal sequence and at least 1, at least 2, or 3 substitutions fromthe C-terminal sequence of the IFN-α14 are made to provide residues fromIFN-α10 to the hybrid at those amino acid positions which have aminoacids that are not shared/common between IFN-α10 and IFN-α14.

In embodiments, the hybrid comprises or consists of an amino acidsequence SEQ ID NO: 1 or a functionally active fragment or variantthereof.

In certain embodiments, the method includes a step of administering tothe subject a therapeutically effective amount of a vaccine compositionfor treatment or prophylaxis of the condition where an enhancement of aTh1-mediated immune response and suppression of a Th2/Th17-mediatedimmune response are desired. The vaccine composition may be administeredsequentially, separately or simultaneously with the at least oneinterferon alpha subtype.

By functionally active is meant an IL-α10 IL-α14 hybrid peptidecomprising the primary interferon binding sites of IFN-α10 and IFN-α14wherein the administration of peptide to a subject or expression ofpeptide in a subject promotes enhancement of Th1 mediated immuneresponse and suppression of a Th2/Th17 mediated immune response.Further, functional activity may be indicated by the ability of a hybridpeptide to enhance a Th1 mediated immune response and to suppress aTh2/Th17 mediated response.

A fragment can comprise at least 50, preferably 100 and more preferably150 or greater contiguous amino acids from SEQ ID NO: 1 and which isfunctionally active. Suitably, a fragment may be determined using, forexample, C-terminal serial deletion of cDNA such as SEQ ID NO: 2 or SEQID NO: 3. Said deletion constructs may then be cloned into suitableplasmids. The activity of these deletion mutants may then be tested forbiological activity as described herein.

By variant is meant an amino acid sequence which is at least 70%homologous to SEQ ID NO: 1, more preferably at least 80% homologous toSEQ ID NO: 1, more preferably at least 90% homologous to SEQ ID NO: 1,even more preferably at least 95% homologous to SEQ ID NO: 1, even morepreferably at least 96% homologous to SEQ ID NO: 1, even more preferablyat least 97% homologous to SEQ ID NO: 1 and most preferably at least 98%homology with SEQ ID NO: 1. A variant encompasses a polypeptide sequenceof SEQ ID NO: 1 which includes substitution of amino acids, especially asubstitution(s) which is/are known for having a high probability of notleading to any significant modification of the biological activity orconfiguration, or folding, of the protein. These substitutions,typically known as conserved substitutions, are known in the art. Forexample the group of arginine, lysine and histidine are knowninterchangeable basic amino acids. Suitably, in embodiments amino acidsof the same charge, size or hydrophobicity may be substituted with eachother. Suitably, any substitution may be selected based on analysis ofamino acid sequence alignments of interferon alpha subtypes to provideamino acid substitutions to amino acids which are present in other alphasubtypes at similar or identical positions when the sequences arealigned. Hybrids, and variants and fragments thereof may be generatedusing suitable molecular biology methods as known in the art.

In certain embodiments, the vaccine composition comprises at least oneantigen. In certain embodiments, the vaccine composition comprises atleast one allergen capable of mediating a Th2/Th17 immune response, forexample, a food allergen.

In aspects and embodiments of the invention the antigen can be a tumourantigen, for example a tumour specific antigen or a tumour associatedantigen, in particular a tumour antigen can be of a hepatic carcinoma,lung cancer, in particular non-small cell lung cancer, ovarian cancer,breast cancer, skin cancer, melanoma or of a genitourinary cancer. Inparticular an antigen of a genitourinary cancer can include an antigenfrom a prostate cancer, renal cell carcinoma, or bladder cancer.Suitably, an antigen may be a tumour specific antigen or tumourassociated antigen provided in an existing cancer vaccine in use ordevelopment which would benefit from an adjuvant that enhances T-cellimmunity, in particular that enhances a Th1 response or provides anenhancement of a Th1 mediated immune response and suppression of aTh2/Th17-mediated immune response. Suitably a tumour specific ortumour-associated antigen may be obtained from a tumour of a subject tobe treated. In embodiments only a tumour-associated antigen can be used.

In embodiments a tumour antigen, in particular an associated antigen maybe an antigen for a prostate cancer antigen, in particularprostate-specific antigen. Suitably a method of providing a prostatespecific antigen or a prostate cancer antigen with the interferon-alphasubtypes of the invention maybe used to treat prostate cancer,specifically castration-resistant prostate cancer.

As will be appreciated by a physician, the subjects who will benefitmost from such treatments may be those with minimal disease, as theremay be less chance of increasing tumour suppression of the immunesystem, additionally or alternatively such treatments may benefitsubjects with advanced disease who may have significant tumour immunesuppression and may benefit more from the use of vaccines in combinationwith other forms of treatment. Suitably the use of vaccines includingtumour antigens, in particular tumour associated antigen may be incombination with other forms of immunotherapy, for example Sunitinib(Sutent by Pfizer) a tyrosine kinase inhibitor.

In embodiments specific tumour antigens, in particular tumour-associatedantigens may be selected from the antigens utilised in the prostatecancer vaccines TroVax and Prostvac.

In embodiments a tumour antigen, in particular a tumour-associatedantigen can be selected from renal cell carcinoma. Suitably a tumourantigen, for example a tumour-associated antigen for renal cellcarcinoma may be selected from a heat shock protein or proteins of renaltumour cell lysates, in particular the antigen used in the potentialvaccine MVA-5T4.

Suitably a tumour antigen may be MUC1 from melanoma.

In embodiments, a tumour antigen, for example a tumour-associatedantigen can be selected from bladder cancer. Suitably atumour-associated antigen may be selected from Bacille Calmette-Guerin(BCG) vaccine, human leukocyte antigen-A*2402 restricted epitopepeptides, immucin peptide (a 21mer synthetic vaccine composed of theentire signal peptide of the MUCI protein) human chorionicgonadotropin-colony stimulating factor, or human chorionicgonadotropin-beta.

In certain embodiments, the method therefore includes a step ofadministering to the subject a therapeutically effective amount of atleast one allergen capable of mediating a Th2/Th17 immune response, forexample, a food allergen or tumour antigen, for example a tumourassociated antigen. The allergen maybe administered sequentially,separately or simultaneously with the at least one interferon alphasubtype.

Typically, the subject is a mammal, in particular a human. In certainembodiments, the subject can be suffering from a condition where anenhancement of a Th1-mediated immune response and suppression of aTh2/Th17-mediated immune response are desired.

According to a second aspect of the present invention, there is providedat least one interferon alpha subtype which is a hybrid of IFN-α10 andIFN-α14 wherein the hybrid comprises the primary interferon bindingsites of IFN-α10 and IFN-α14, in particular wherein the IFN-α10-IFN-α14hybrid sequence comprises at least one mutation selected from aminoacids at positions 94, 101, 102, 109 or 144, preferably at least twomutations selected from amino acids at positions 94, 101, 102, 109 or144, more preferably at least three mutations selected from amino acidsat positions 94, 101, 102, 109 or 144, more preferably at least fourmutations selected from amino acids at positions 94, 101, 102, 109 or144 or more preferably at least five mutations selected from amino acidsat positions 94, 101, 102, 109 or 144, in particular SEQ ID NO:1 or afragment or variant thereof for use in the treatment and/or prophylaxisof a condition where an enhancement of a Th1-mediated immune responseand suppression of a Th2/Th17-mediated immune response are desired.

In certain embodiments, the at least one interferon alpha subtype, inparticular a hybrid IFN-α10 and IFN-α14 subtype, for example SEQ ID NO:1, as described herein is provided for simultaneous, separate orsequential administration with a vaccine composition for treatment orprophylaxis of the condition where an enhancement of a Th1-mediatedimmune response and suppression of a Th2/Th17-mediated immune responseare desired. In certain embodiments, the at least one interferon alphasubtype is provided for simultaneous, separate or sequentialadministration with at least one allergen capable of mediating aTh2/Th17 immune response there against, for example, a food allergen, ora tumour antigen, in particular a tumour-associated antigen.

According to a third aspect of the present invention, there is provideduse of at least one interferon alpha subtype which is a hybrid ofIFN-α10 and IFN-α14 wherein the hybrid comprises the primary interferonbinding sites of IFN-α10 and IFN-α14, in particular wherein theIFN-α10-IFN-α14 hybrid sequence comprises at least one mutation selectedfrom amino acids at positions 94, 101, 102, 109 or 144, preferably atleast two mutations selected from amino acids at positions 94, 101, 102,109 or 144, more preferably at least three mutations selected from aminoacids at positions 94, 101, 102, 109 or 144, more preferably at leastfour mutations selected from amino acids at positions 94, 101, 102, 109or 144 or more preferably at least five mutations selected from aminoacids at positions 94, 101, 102, 109 or 144, in particular wherein thehybrid can be SEQ ID No:1 or a variant or fragment thereof in thepreparation of a medicament for the treatment and/or prophylaxis of acondition where an enhancement of a Th1-mediated immune response andsuppression of a Th2/Th17-mediated immune response are desired.

In certain embodiments, the at least one interferon alpha subtype isprovided for simultaneous, separate or sequential administration with avaccine composition for treatment or prophylaxis of the condition wherean enhancement of a Th1-mediated immune response and suppression of aTh2/Th17-mediated immune response are desired. In certain embodiments,the at least one interferon alpha subtype is provided for simultaneous,separate or sequential administration with at least one allergen capableof mediating a Th2/Th17 immune response there against, for example, afood allergen, or a tumour antigen, in particular a tumour associatedantigen.

According to a further aspect of the present invention, there isprovided a composition comprising:

-   -   (i) a vaccine for treatment or prophylaxis of a condition where        an enhancement of a Th1-mediated immune response and suppression        of a Th2/Th17-mediated immune response are desired; and    -   (ii) at least one interferon alpha subtype which is a hybrid of        IFN-α10 and IFN-α14, in particular wherein the hybrid comprises        the primary interferon binding sites of IFN-α10 and IFN-α14, in        particular wherein the IFN-α10-IFN-α14 hybrid sequence comprises        at least one mutation selected from amino acids at positions 94,        101, 102, 109 or 144, preferably at least two mutations selected        from amino acids at positions 94, 101, 102, 109 or 144, more        preferably at least three mutations selected from amino acids at        positions 94, 101, 102, 109 or 144, more preferably at least        four mutations selected from amino acids at positions 94, 101,        102, 109 or 144 or more preferably at least five mutations        selected from amino acids at positions 94, 101, 102, 109 or 144,        in particular wherein the hybrid can be SEQ ID NO:1 or a variant        or fragment, as described herein.

In certain embodiments, the vaccine comprises at least one allergencapable of mediating a Th2/Th17 immune response, for example, a foodallergen or a tumour antigen, in particular a tumour-associated antigen.

A further aspect of the present invention provides a pharmaceuticalcomposition for enhancement of a Th1 mediated immune response andsuppression of a Th2/Th17-mediated immune response, wherein thecomposition comprises a vaccine for treatment or prophylaxis of acondition where an enhancement of a Th1-mediated immune response andsuppression of a Th2/Th17-mediated immune response are desired and atleast one interferon alpha subtype which is a hybrid of IFN-α10 andIFN-α14, in particular wherein the hybrid comprises the primaryinterferon binding sites of IFN-α10 and IFN-α14, in particular whereinthe IFN-α10-IFN-α14 hybrid sequence comprises at least one mutationselected from amino acids at positions 94, 101, 102, 109 or 144,preferably at least two mutations selected from amino acids at positions94, 101, 102, 109 or 144, more preferably at least three mutationsselected from amino acids at positions 94, 101, 102, 109 or 144, morepreferably at least four mutations selected from amino acids atpositions 94, 101, 102, 109 or 144 or more preferably at least fivemutations selected from amino acids at positions 94, 101, 102, 109 or144, in particular wherein the hybrid can be SEQ ID NO:1 or a fragmentor variant thereof along with a pharmaceutically acceptable excipient,diluent or carrier.

In certain embodiments, the vaccine comprises at least one allergencapable of mediating a Th2/Th17 immune response, for example, a foodallergen or tumour antigen, in particular a tumour-associated antigen.

In a further aspect, the present invention extends to improvements inthe efficacy of vaccines, for example, anti-allergy or allergic diseasevaccines or tumour or cancer vaccines, in particular genitourinarycancer vaccines, for example prostate cancer, renal cancer and orbladder cancer. A composition which comprises a vaccine for treatment orprophylaxis of a condition where an enhancement of a Th1-mediated immuneresponse and suppression of a Th2/Th17-mediated immune response aredesired, such as at least one allergen capable of mediating a Th2/Th17immune response, and at least one interferon alpha subtype which is ahybrid of IFN-α10 and IFN-α14 in particular wherein the hybrid comprisesthe primary interferon binding sites of IFN-α10 and IFN-α14, inparticular wherein the IFN-α10-IFN-α14 hybrid sequence comprises atleast one mutation selected from amino acids at positions 94, 101, 102,109 or 144, preferably at least two mutations selected from amino acidsat positions 94, 101, 102, 109 or 144, more preferably at least threemutations selected from amino acids at positions 94, 101, 102, 109 or144, more preferably at least four mutations selected from amino acidsat positions 94, 101, 102, 109 or 144 or more preferably at least fivemutations selected from amino acids at positions 94, 101, 102, 109 or144, in particular SEQ ID NO:1 or a variant or fragment thereof, hasbeen surprisingly identified by the inventor as providing anunexpectedly efficacious composition for the treatment and/orprophylaxis of diseases, such as allergy or associated allergicdiseases.

Accordingly, a further aspect of the present invention provides avaccine composition comprising;

(i) a vaccine for treatment or prophylaxis of a condition where anenhancement of a Th1-mediated immune response and suppression of aTh2/Th17-mediated immune response are desired; and(ii) at least one interferon alpha subtype which is a hybrid of IFN-α10and IFN-α14 in particular wherein the hybrid comprises the primaryinterferon binding sites of IFN-α10 and IFN-α14, in particular whereinthe IFN-α10-IFN-α14 hybrid sequence comprises at least one mutationselected from amino acids at positions 94, 101, 102, 109 or 144,preferably at least two mutations selected from amino acids at positions94, 101, 102, 109 or 144, more preferably at least three mutationsselected from amino acids at positions 94, 101, 102, 109 or 144, morepreferably at least four mutations selected from amino acids atpositions 94, 101, 102, 109 or 144 or more preferably at least fivemutations selected from amino acids at positions 94, 101, 102, 109 or144, in particular can be SEQ ID NO:1 or a variant or fragment thereof.

In certain embodiments, the vaccine comprises at least one allergencapable of mediating a Th2/Th17 immune response, for example, a foodallergen or a tumour antigen, in particular a tumour-associated antigen.

A further aspect of the present invention provides a vaccine compositionfor use in the treatment and/or prophylaxis of allergy or cancer, inparticular genitourinary cancer, for example prostate cancer, renalcancer or bladder cancer, where an enhancement of a Th1-mediated immuneresponse and the suppression of a Th2/Th17-mediated immune response aredesired, said vaccine composition comprising;

(i) at least one allergen capable of mediating a Th2/Th17 immuneresponse; and(ii) at least one interferon alpha subtype which is a hybrid IFN-α10 andIFN-α14 subtype, in particular wherein the hybrid comprises the primaryinterferon binding sites of IFN-α10 and IFN-α14, in particular whereinthe IFN-α10-IFN-α14 hybrid sequence comprises at least one mutationselected from amino acids at positions 94, 101, 102, 109 or 144,preferably at least two mutations selected from amino acids at positions94, 101, 102, 109 or 144, more preferably at least three mutationsselected from amino acids at positions 94, 101, 102, 109 or 144, morepreferably at least four mutations selected from amino acids atpositions 94, 101, 102, 109 or 144 or more preferably at least fivemutations selected from amino acids at positions 94, 101, 102, 109 or144, in particular wherein the hybrid can be SEQ ID NO:1 or a variant orfragment, as described herein.

A further aspect of the present invention provides for the use of avaccine composition comprising at least one allergen capable ofmediating a Th2/Th17 immune response and at least one interferon alphasubtype which is a hybrid of IFN-α10 and IFN-α14 in particular whereinthe hybrid comprises the primary interferon binding sites of IFN-α10 andIFN-α14, in particular wherein the IFN-α10-IFN-α14 hybrid sequencecomprises at least one mutation selected from amino acids at positions94, 101, 102, 109 or 144, preferably at least two mutations selectedfrom amino acids at positions 94, 101, 102, 109 or 144, more preferablyat least three mutations selected from amino acids at positions 94, 101,102, 109 or 144, more preferably at least four mutations selected fromamino acids at positions 94, 101, 102, 109 or 144 or more preferably atleast five mutations selected from amino acids at positions 94, 101,102, 109 or 144, in particular wherein the hybrid can be SEQ ID NO:1 ora variant or fragment thereof, in the preparation of a medicament forthe treatment and/or prophylaxis of allergy or associated allergicdiseases, or cancer, in particular genitourinary cancer, for exampleprostate cancer, renal cancer or bladder cancer.

A further aspect of the present invention provides a method for thetreatment and/or prophylaxis of allergy or associated allergic diseasesor of cancer, in particular genitourinary cancer for example prostatecancer, renal cancer or bladder cancer the method comprising the stepof:

-   -   (i) administering a therapeutically effective amount of a        vaccine composition or an immunogenic composition which        comprises at least one allergen capable of mediating a Th2/Th17        immune response and at least one interferon alpha subtype which        is a hybrid of IFN-α10 and IFN-α14, in particular wherein the        hybrid comprises the primary interferon binding sites of IFN-α10        and IFN-α14, in particular wherein the IFN-α10-IFN-α14 hybrid        sequence comprises at least one mutation selected from amino        acids at positions 94, 101, 102, 109 or 144, preferably at least        two mutations selected from amino acids at positions 94, 101,        102, 109 or 144, more preferably at least three mutations        selected from amino acids at positions 94, 101, 102, 109 or 144,        more preferably at least four mutations selected from amino        acids at positions 94, 101, 102, 109 or 144 or more preferably        at least five mutations selected from amino acids at positions        94, 101, 102, 109 or 144, in particular wherein the hybrid can        be SEQ ID NO:1 or a fragment or variant thereof to a subject in        need thereof.

According to a further aspect of the present invention, there isprovided a method for the treatment and/or prophylaxis of a conditionmediated by enhanced expression of IL-17, said method comprising thestep of:

-   -   (i) administering to a subject in need thereof a therapeutically        effective amount of at least one interferon alpha subtype which        is a hybrid of IFN-α10 and IFN-α14 in particular wherein the        hybrid comprises the primary interferon binding sites of IFN-α10        and IFN-α14, in particular wherein the IFN-α10-IFN-α14 hybrid        sequence comprises at least one mutation selected from amino        acids at positions 94, 101, 102, 109 or 144, preferably at least        two mutations selected from amino acids at positions 94, 101,        102, 109 or 144, more preferably at least three mutations        selected from amino acids at positions 94, 101, 102, 109 or 144,        more preferably at least four mutations selected from amino        acids at positions 94, 101, 102, 109 or 144 or more preferably        at least five mutations selected from amino acids at positions        94, 101, 102, 109 or 144, in particular SEQ ID NO:1 or a        fragment or variant thereof.

According to a further aspect of the present invention, there isprovided at least one interferon alpha subtype comprising or consistingof an IFN-α10 and IFN-α14 hybrid in particular wherein the hybridcomprises the primary interferon binding sites of IFN-α10 and IFN-α14,in particular wherein the IFN-α10-IFN-α14 hybrid sequence comprises atleast one mutation selected from amino acids at positions 94, 101, 102,109 or 144, preferably at least two mutations selected from amino acidsat positions 94, 101, 102, 109 or 144, more preferably at least threemutations selected from amino acids at positions 94, 101, 102, 109 or144, more preferably at least four mutations selected from amino acidsat positions 94, 101, 102, 109 or 144 or more preferably at least fivemutations selected from amino acids at positions 94, 101, 102, 109 or144, in particular SEQ ID NO:1 or a variant or fragment thereof for usein the treatment and/or prophylaxis of a condition mediated by enhancedexpression of IL-17.

Suitably, in aspects and embodiments of the invention, the hybrid maycomprise or consist of the amino acid sequence of SEQ ID NO:1.

According to a further aspect of the present invention, there isprovided use of at least one interferon alpha subtype IFN-α10 andIFN-α14 hybrid in particular wherein the hybrid comprises the primaryinterferon binding sites of IFN-α10 and IFN-α14, in particular whereinthe IFN-α10-IFN-α14 hybrid sequence comprises at least one mutationselected from amino acids at positions 94, 101, 102, 109 or 144,preferably at least two mutations selected from amino acids at positions94, 101, 102, 109 or 144, more preferably at least three mutationsselected from amino acids at positions 94, 101, 102, 109 or 144, morepreferably at least four mutations selected from amino acids atpositions 94, 101, 102, 109 or 144 or more preferably at least fivemutations selected from amino acids at positions 94, 101, 102, 109 or144, in particular SEQ ID NO:1 or a variant or fragment thereof, in thepreparation of a medicament for the treatment and/or prophylaxis of acondition mediated by enhanced expression of IL-17.

According to a further aspect of the present invention, there isprovided a method for modulating an immune response, said methodcomprising the step of:

-   -   (i) administering to a subject in need thereof a therapeutically        effective amount of at least one interferon alpha subtype        IFN-α10 and IFN-α14 hybrid, wherein the hybrid comprises the        primary interferon binding sites of IFN-α10 and IFN-α14, in        particular wherein the IFN-α10-IFN-α14 hybrid sequence comprises        at least one mutation selected from amino acids at positions 94,        101, 102, 109 or 144, preferably at least two mutations selected        from amino acids at positions 94, 101, 102, 109 or 144, more        preferably at least three mutations selected from amino acids at        positions 94, 101, 102, 109 or 144, more preferably at least        four mutations selected from amino acids at positions 94, 101,        102, 109 or 144 or more preferably at least five mutations        selected from amino acids at positions 94, 101, 102, 109 or 144,        and in particular can be SEQ ID NO:1 or a variant or fragment        thereof.

Suitably, in aspects and embodiments of the invention, theadministration or use of at least one interferon alpha subtypecomprising or consisting of an IFN-α10 and IFN-α14 hybrid in particularwherein the hybrid comprises the primary interferon binding sites ofIFN-α10 and IFN-α14, in particular wherein the IFN-α10-IFN-a14 hybridsequence comprises at least one mutation selected from amino acids atpositions 94, 101, 102, 109 or 144, preferably at least two mutationsselected from amino acids at positions 94, 101, 102, 109 or 144, morepreferably at least three mutations selected from amino acids atpositions 94, 101, 102, 109 or 144, more preferably at least fourmutations selected from amino acids at positions 94, 101, 102, 109 or144 or more preferably at least five mutations selected from amino acidsat positions 94, 101, 102, 109 or 144, in particular SEQ ID NO:1 or avariant or fragment thereof results in the full or partial inhibition ofIL-17 and/or the full or partial activation of IFN-γ.

According to a further aspect of the present invention, there isprovided at least one interferon alpha subtype IFN-α10 and IFN-α14hybrid, wherein the hybrid comprises the primary interferon bindingsites of IFN-α10 and IFN-α14, in particular wherein the IFN-α10-IFN-α14hybrid sequence comprises at least one mutation selected from aminoacids at positions 94, 101, 102, 109 or 144, preferably at least twomutations selected from amino acids at positions 94, 101, 102, 109 or144, more preferably at least three mutations selected from amino acidsat positions 94, 101, 102, 109 or 144, more preferably at least fourmutations selected from amino acids at positions 94, 101, 102, 109 or144 or more preferably at least five mutations selected from amino acidsat positions 94, 101, 102, 109 or 144, and in particular can be SEQ IDNO:1 or a variant or fragment thereof for use in modulating an immuneresponse.

According to a further aspect of the present invention, there isprovided use of at least one interferon alpha subtype hybrid IFN-α10 andIFN-α14 subtype, wherein the hybrid comprises the primary interferonbinding sites of IFN-α10 and IFN-α14, in particular wherein theIFN-α10-IFN-α14 hybrid sequence comprises at least one mutation selectedfrom amino acids at positions 94, 101, 102, 109 or 144, preferably atleast two mutations selected from amino acids at positions 94, 101, 102,109 or 144, more preferably at least three mutations selected from aminoacids at positions 94, 101, 102, 109 or 144, more preferably at leastfour mutations selected from amino acids at positions 94, 101, 102, 109or 144 or more preferably at least five mutations selected from aminoacids at positions 94, 101, 102, 109 or 144, and in particular SEQ IDNO:1 or a variant or fragment thereof in the preparation of a medicamentfor modulating an immune response.

In certain embodiments of the aspects of the invention outlined above,the at least one interferon alpha subtype is provided for simultaneous,separate or sequential administration with a vaccine for treatment orprophylaxis of the condition where an enhancement of a Th1-mediatedimmune response and suppression of a Th2/Th17-mediated immune responseare desired, for example, a vaccine for the treatment or prophylaxis ofa condition mediated by enhanced expression of IL-17, e.g. aninflammatory disease or condition or an autoimmune disease, such asinflammatory bowel disease (IBD), ulcerative colitis (UC) or Crohn'sdisease (CD), cancer, suitably hepatic cancer, lung cancer, inparticular non-small cell lung cancer, ovarian cancer, breast cancer,skin cancer, melanoma or genitourinary cancer, in particulargenitourinary cancer, for example prostate cancer, renal cancer orbladder cancer. In certain embodiments, the vaccine compositioncomprises at least one antigen. In certain embodiments, the vaccinecomprises at least one allergen capable of mediating a Th2/Th17 immuneresponse there against, for example, a food allergen.

In certain embodiments the antigen can be a tumour antigen in particulara tumour specific and/or a tumour-associated antigen.

In certain embodiments of the aspects of the invention outlined above,the at least one IFN-α subtype comprises, consists of or is an IFN-α10IFN-α14 hybrid such as a fusion protein, or recombinant protein or thelike which includes the primary interferon receptor binding sites ofIFN-α10 and IFN-α14, and in particular which comprises or consists ofthe amino acid sequence SEQ ID NO:1 or a variant or fragment thereof. Inembodiments the IFN-α10 IFN-α14 hybrid can be glycosylated. Suitably theIFN-α10 IFN-α14 hybrid can be glycosylated in a similar fashion toIFN-α14.

In certain embodiments of the aspects of the invention outlined above,the at least one allergen is at least one food allergen or a tumourspecific or tumour-associated tumour allergen, for example a prostatecancer allergen, a renal cancer allergen and or bladder cancer allergen.In certain embodiments, the at least one allergen is a dietary allergensuch as food, an environmental allergen such as the venom of stinginginsects, or a medication.

In a further aspect of the invention there is provided a recombinantpolypeptide comprising or consisting of SEQ ID NO:1 or a fragment orvariant thereof. Nucleic acid sequences derived from the amino acidsequence SEQ ID NO:1 are provided as SEQ ID NO:2. These nucleic acidsequences can form additional aspects to the invention.

In certain embodiments of the aspects of the invention outlined above,the at least one food allergen is selected from the group consisting of,but not limited to, corn, garlic, oats, coffee, chocolate, pickle, wheator gluten and their products or derivatives which include durum wheat,spelt (triticum spelta), kamut (triticum poloncium), couscous, bran,wheat bran, wheat germ, wheat gluten, farina, rusk, semolina, durumwheat semolina, flour, wholewheat flour, wheat flour, wheat starch,starch, modified starch, hydrolysed starch, food starch, edible starch,vegetable starch, vegetable gum, vegetable protein, cereal filler,cereal binder, cereal protein; tree nuts (including almonds, cashews,macademia, walnut and brazil nuts); seeds, including sesame, sunflowerand poppy seeds; dairy derived antigens, such as milk or milkderivatives, including cheese and yoghurt; fish or shellfish or theirderivatives, including from the mollusc phylum (gastropod class: snailsand abalone; bivalve class: clam, mussel and oyster; cephalopod class:octopus, squid and scallop), arthropod phylum (crustacean family: crab,lobster, shrimp, prawn and crayfish) or chordate phylum (cartilaginousfamily: ray and shark; bony fish: cod, salmon and tuna); eggs or eggderivatives; monosodium glutamate (MSG); sulphites or sulphur dioxide;legume allergies to the leguminosae family, which includes peanut, soya(soybean or soya derivatives), bean seeds, peas, green beans, lentils,carob and liquorice; other vegetable allergies such as potato; fruitallergies to the rosaceae family, which includes apple, pear, cherry,peach and plum; fruit allergies to the cucurbitaceae family, whichincludes cucumber, melon, watermelon, zucchini and pumpkin; and otherfruit allergies such as those developed against kiwi, banana, avocado,tomatoes, strawberries and raspberries.

In certain embodiments, the vaccine or vaccine composition can be avaccine composition for the treatment or prophylaxis of a conditionmediated by enhanced expression of IL-17, e.g. an inflammatory diseaseor condition or an autoimmune disease, such as inflammatory boweldisease (IBD), ulcerative colitis (UC) or Crohn's disease (CD), orcancer, suitably hepatic cancer, lung cancer, non-small cell lungcancer, ovarian cancer, breast cancer, skin cancer, melanoma orgenitourinary cancer, in particular genitourinary cancer, for exampleprostate cancer, renal cancer or bladder cancer. In certain embodiments,the vaccine or vaccine composition can be a vaccine composition for thetreatment or prophylaxis of an inflammatory disease or condition or anautoimmune disease, such as inflammatory bowel disease (IBD), ulcerativecolitis (UC) or Crohn's disease (CD).

In certain embodiments of the aspects of the invention outlined above,the condition where an enhancement of a Th1-mediated immune response andthe suppression of a Th2/Th17-mediated immune response are desired canbe a condition mediated by enhanced expression of IL-17, e.g. aninflammatory disease or condition or an autoimmune disease, such asinflammatory bowel disease (IBD), ulcerative colitis (UC) or Crohn'sdisease (CD).

In certain embodiments of the aspects of the invention outlined above,the condition where an enhancement of a Th1-mediated immune response andthe suppression of a Th2/Th17-mediated immune response are desired canbe an inflammatory disease, in particular an inflammatory disease whichis mediated by an exaggerated or overactive Th17 immune response. Incertain embodiments of the aspects of the invention outlined above, thecondition where an enhancement of a Th1-mediated immune response and thesuppression of a Th2/Th17-mediated immune response are desired can be anautoimmune disease, in particular an autoimmune disease which ismediated by an exaggerated or overactive Th17 immune response. Forexample, in certain embodiments the condition can be inflammatory boweldisease (IBD), such as ulcerative colitis (UC) or Crohn's disease (CD).In certain embodiments, the condition can be selected from the groupconsisting of asthma, allergic rhinitis, atopic dermatitis and foodallergy. In certain embodiments, the condition is cancer, in particulara genitourinary cancer, in particular prostate cancer, bladder cancer orrenal cancer.

In certain embodiments of the aspects of the invention outlined above,the condition where an enhancement of a Th1-mediated immune response andthe suppression of a Th2/Th17-mediated immune response are desired is anallergy or associated allergic diseases and conditions caused thereby,or cancer wherein an immune response is desired against atumour-associated antigen, in particular a tumour associated antigen ofprostate cancer, renal cancer or bladder caner. In particular, incertain embodiments the condition is a food allergy including foodassociated or derived allergies and associated allergic diseases andconditions caused thereby.

In certain embodiments, the food allergy associated allergic diseases orconditions include, but are not limited to, milk/dairy allergy,including Heiner syndrome, egg allergy, soya allergy, fish (shellfish)allergy, peanut and tree nut allergy, sesame and other seed allergy,wheat and grains allergy, fruit and vegetable allergy, caffeine allergy,oral allergy syndrome, alcohol allergy, pollen food allergy syndrome,eosinophilic gastroenteritis, IgE mediated gastrointestinal food allergyand C1 esterase deficiency.

In certain embodiments of the present invention, the method ofadministration is oral administration. In certain embodiments, themethod of administration is sublingual or buccal administration. Incertain embodiments, the method of administration involves placing alozenge under the patient's tongue. In certain embodiments, the route ofadministration is ocular or by means of introduction into the nasalcavity, by way of nasal administration. Also it may be introduced byoral administration (swallowing) of a capsule or similar device into thesmall intestine/duodenum such that the capsule does not dissolve in thestomach, but bypasses same and delivers/releases the interferon alphasubtype only into the small intestine/duodenum.

DETAILED DESCRIPTION OF THE INVENTION

The inventor of the present invention has surprisingly discovered thatadministering an IFN-α subtype which is a hybrid of IFN-α10 and IFN-α14subtypes, for example SEQ ID NO:1, as described herein results in theenhancement of a Th1 T cell mediated immune response and the suppressionof a Th2/Th17 T cell mediated immune response and can therefore skew theimmune response towards a cell-mediated (Th1) path, whilstsimultaneously suppressing the allergic (Th2/Th17) response.Surprisingly, this effect is enhanced when the IFN-α subtype isadministered orally.

In particular, the inventor discovered that IFN-α10-IFN-α14 hybrids thatcontain sequences characteristic of the IFN-α10 and IFN-α14 subtypebinding sites that are not based on a consensus sequence of all 12 IFN-αsubtypes resulted in a protein with higher affinity binding sites forthe two interferon receptors, IFNR1 and IFNR2. This finding can beapplied to provide an improved method and improved adjuvant compositionfor treating and/or preventing conditions where the enhancement of a Th1T cell mediated immune response and/or the suppression of a Th2/Th17 Tcell mediated immune response are desired, for example, inflammatory,autoimmune or allergy conditions, or cancer (including malignantconditions), in particular genitourinary cancers, in particular prostatecancer, renal cancer or bladder cancer. In particular, the hybrid ofIFN-α10 and IFN-α14, in particular wherein the hybrid comprises theprimary interferon binding sites of IFN-α10 and IFN-α14, in particularwherein the IFN-α10-IFN-α14 hybrid sequence comprises at least onemutation selected from amino acids at positions 94, 101, 102, 109 or144, preferably at least two mutations selected from amino acids atpositions 94, 101, 102, 109 or 144, more preferably at least threemutations selected from amino acids at positions 94, 101, 102, 109 or144, more preferably at least four mutations selected from amino acidsat positions 94, 101, 102, 109 or 144 or more preferably at least fivemutations selected from amino acids at positions 94, 101, 102, 109 or144, and in particular SEQ ID NO:1 or a fragment or variant thereof maybe used as an adjuvant in vaccines to boost immune response to antigensand direct the immune response towards a Th1 immune response.

The inventor has also discovered that a combination of a vaccinecomposition or a food or tumour specific or tumour-associated antigenallergen which is capable of mediating a Th2/Th17 immune response and anIFN-α subtype which is a hybrid of IFN-α10 and IFN-α14, in particular ahybrid comprising the primary interferon binding sites of IFN-α10 andIFN-α14, in particular wherein the IFN-α10-IFN-α14 hybrid sequencecomprises at least one mutation selected from amino acids at positions94, 101, 102, 109 or 144, preferably at least two mutations selectedfrom amino acids at positions 94, 101, 102, 109 or 144, more preferablyat least three mutations selected from amino acids at positions 94, 101,102, 109 or 144, more preferably at least four mutations selected fromamino acids at positions 94, 101, 102, 109 or 144 or more preferably atleast five mutations selected from amino acids at positions 94, 101,102, 109 or 144, and in particular SEQ ID NO:1 or a fragment or variantthereof can result in the activation of a Th1 T cell mediated immuneresponse and the suppression of a Th2/Th17 T cell mediated immuneresponse.

Tumour progression in normal immunocompetent subjects may reflect afailure of the immune system to recognize the tumour antigens or asubversion of the anti-tumour immune response through induction andactivation of regulatory T cells. In subjects with hepaticchoriocarcinoma (HCC) studies of IL-17α cells have suggested a potentialpro-tumour role for IL-17. Increased IL-17 producing cell density withinthe tumours of HCC patients correlates with both microvessel density andpoor prognosis. Further, in subjects with non-small cell lung andovarian cancer, higher levels of IL-17 within the tumour correlated withhigher blood vessel density and shorter survival. Additionally IL-17 hasbeen suggested to have pro-angiogenic roles and this has not beenrestricted to particular cell populations. Moreover, it has been shownthat IL-17A or IL-17A producing cells are elevated in the environment ofbreast tumours and correlate with poor prognosis.

Isolation of tumour infiltrating lymphocytes (TILS) from breast cancerbiopsies revealed these cells secreted significant amounts of IL-17A,and that recombinant IL-17A recruits the MAPK pathway by upregulatingphosphorylated ERK 1/2 in human breast cancer lines thereby promotingproliferation and resistance to conventional chemotherapeutic agentssuch as Docetaxel. IL-17A has also been indicated to stimulate migrationand invasion of breast cancer cells. Importantly IL-17A-neutralizingantibodies abrogated these effects, demonstrating the pathophysiologicalrole of IL-17A as a potential therapeutic target for breast cancer. Theinventor has surprisingly discovered that administration of the novelIFN-α10-IFN-α14 hybrid result in a greater reduction of IL-17 comparedto previous IFN-α10-IFN-α14 hybrid. The inventor has discovered thatadministration of the novel IFN-α10-IFN-α14 hybrid results in a 10%,preferably a 20%, preferably a 30%, preferably a 40% and more preferablya 50% greater reduction of IL-17 compared to previous IFN-α10-IFN-α14hybrid. The determination by the inventor or means thus to activate aTh1 T cell mediated immune response and suppress a Th2/Th17 T cellmediated immune response is therefore significant and of utility incancer. Thus, the present invention may be used for the treatment andprophylaxis of any known cancerous or malignant condition.

In addition, the inventor has discovered that the administration or useof at least one interferon alpha subtype comprising or consisting of anIFN-α10 and IFN-α14 hybrid in particular wherein the hybrid comprisesthe primary interferon binding sites of IFN-α10 and IFN-α14, inparticular wherein the IFN-α10-IFN-α14 hybrid sequence comprises atleast one mutation selected from amino acids at positions 94, 101, 102,109 or 144, preferably at least two mutations selected from amino acidsat positions 94, 101, 102, 109 or 144, more preferably at least threemutations selected from amino acids at positions 94, 101, 102, 109 or144, more preferably at least four mutations selected from amino acidsat positions 94, 101, 102, 109 or 144 or more preferably at least fivemutations selected from amino acids at positions 94, 101, 102, 109 or144, in particular SEQ ID NO:1 or a variant or fragment thereof resultsin the full or partial inhibition of IL-17 and/or the full or partialactivation of IFN-γ.

Moreover, the inventor has surprisingly discovered that orallyadministering the antigen and IFN-α subtype which is a hybrid of IFN-α10and IFN-α14 in particular a hybrid comprising the primary interferonbinding sites of IFN-α10 and IFN-α14, in particular wherein theIFN-α10-IFN-α14 hybrid sequence comprises at least one mutation selectedfrom amino acids at positions 94, 101, 102, 109 or 144, preferably atleast two mutations selected from amino acids at positions 94, 101, 102,109 or 144, more preferably at least three mutations selected from aminoacids at positions 94, 101, 102, 109 or 144, more preferably at leastfour mutations selected from amino acids at positions 94, 101, 102, 109or 144 or more preferably at least five mutations selected from aminoacids at positions 94, 101, 102, 109 or 144, and in particular SEQ IDNO:1 or a fragment or variant thereof in combination as discussed hereincan result in the activation of a Th1 T cell mediated immune responseand the suppression of a Th2/Th17 T cell mediated immune response. Astandard flu vaccine was mixed with a low dose of leukocyte-derivedinterferon alpha (LDA1) and orally administered to mice. The inventornoted that without the interferon, a small anti-flu antibody responsewas recorded in mice, which was approximately 50 times less than with aninjected vaccine. With interferon-alpha, the response from the orallydelivered vaccine was exactly the same as the injected vaccine. A seriesof buccal immunisations using a standard protein antigen and twointerferons, LDA1 and an isolated subtype IFN-α14, surprisingly resultedin oral immunisation of mice to which the composition was administered.However, the inventor surprisingly noted that while the LDA1 gave abalanced response, IFN-α14 mediated only a significant humoral response.The production of IgG1 is indicative of a Th2 response (humoralimmunity) and the production of IgG2a is indicative of a Th1 response(cell-mediated immunity).

The inventor, whilst not wishing to be bound by theory, has identifiedthat the oral administration of a food allergen capable of mediating aTh2/Th17 immune response and an interferon alpha subtype which is ahybrid of IFN-α10 and IFN-α14 can skew the immune response towards acell-mediated (Th1) path, whilst simultaneously suppressing the allergic(Th2/Th17) response. Accordingly, the inventor has surprisingly shownthat the co-administration of an allergen such as a food derived antigenthat is causative of allergy or associated allergic diseases in asubject with certain interferon subtypes modulates the resulting immuneresponse and skews it away from the Th2/Th17 response which would havebeen expected to develop against the allergen or antigen. Thissurprising finding provides an unexpected approach to treat or preventallergic responses or diseases which occur in subjects as a result ofallergens such as food-derived allergens or tumour associated antigens.

Definitions Subject

As herein defined, a “subject” includes and encompasses mammals such ashumans, primates and livestock animals (e.g. sheep, pigs, cattle,horses, donkeys); laboratory test animals such as mice, rabbits, ratsand guinea pigs; and companion animals such as dogs and cats.

Treatment/Therapy

The term “treatment” is used herein to refer to any regimen that canbenefit a human or non-human animal. The treatment may be in respect ofany existing inflammatory, autoimmune, allergic or allergy-associatedcondition and the treatment may be prophylactic (preventativetreatment). Treatment may include curative or alleviative effects.Reference herein to “therapeutic” and “prophylactic” treatment is to beconsidered in its broadest context. The term “therapeutic” does notnecessarily imply that a subject is treated until total recovery.Similarly, “prophylactic” does not necessarily mean that the subjectwill not eventually contract a disease condition. Accordingly,therapeutic and/or prophylactic treatment includes amelioration of thesymptoms of a particular allergic condition or preventing or otherwisereducing the risk of developing a particular allergic condition. Theterm “prophylactic” may be considered as reducing the severity or theonset of a particular condition. “Therapeutic” may also reduce theseverity of an existing condition.

Administration

The active ingredients used in the present invention (e.g. vaccine orallergen and IFN-α10, IFN-α14 or a hybrid thereof) in particular ahybrid IFN-α10 and IFN-α14 subtype, for example SEQ ID NO: 1, asdescribed herein can be administered separately to the same subject,optionally sequentially, or can be co-administered simultaneously as apharmaceutical, immunogenic or vaccine composition. In certainembodiments, the vaccine or allergen is co-administered with theinterferon alpha subtype. The pharmaceutical composition will generallycomprise a suitable pharmaceutical excipient, diluent or carrierselected depending on the intended route of administration.

The active ingredients can be administered to a patient in need oftreatment via any suitable route. The precise dose will depend upon anumber of factors, as is discussed below in more detail.

One suitable route of administration is parenterally (includingsubcutaneous, intramuscular, intravenous, by means of, for example adrip patch). Other suitable routes of administration include (but, arenot limited to) oral, ocular, nasal, topical (including buccal andsublingual), infusion, intradermal or administration via oral or nasalinhalation, by means of, for example, a nebuliser or inhaler, or by animplant. Preferable routes of administration include (but, are notlimited to) oral, buccal and sublingual. The compositions of theinvention may also be administered in such a manner that they aredirected to, or released in, specific areas of the gut intestinal tract(such as the small intestine/duodenum). Typically such release willoccur after passage through the stomach, this targeted release beingachievable through the use of coatings and the like.

For intravenous injection, the active ingredient will be in the form ofa parenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as sodium chloride injection, Ringer's injection,Lactated Ringer's injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

The compositions of the present invention for oral administration may bein tablet, capsule, lozenge, powder or liquid form. Oral administrationmay involve placing a lozenge under the tongue of the patient. A tabletmay comprise a solid carrier such as gelatin or an adjuvant. Liquidpharmaceutical compositions generally comprise a liquid carrier such aswater, petroleum, animal or vegetable oils, mineral oil or syntheticoil. Physiological saline solution, dextrose or other saccharidesolution or glycols such as ethylene glycol, propylene glycol orpolyethylene glycol may be included.

The compositions of the present invention may also be administered viamicrospheres, liposomes, other microparticulate delivery systems orsustained release formulations placed in certain tissues includingblood. Suitable examples of sustained release carriers includesemipermeable polymer matrices in the form of shared articles, e.g.suppositories or microcapsules. Examples of the techniques and protocolsmentioned above and other techniques and protocols which may be used inaccordance with the invention can be found in Remington's PharmaceuticalSciences, 18th edition, Gennaro, A. R., Lippincott Williams & Wilkins;20th edition (Dec. 15, 2000) ISBN 0-912734-04-3 and PharmaceuticalDosage Forms and Drug Delivery Systems; Ansel, H. C. et al. 7th EditionISBN 0-683305-72-7, the entire disclosures of which are hereinincorporated by reference.

Pharmaceutical Compositions

As described above, the present invention extends to a pharmaceuticalcomposition for the treatment of inflammatory diseases, autoimmunediseases and allergy such as food allergy and associated allergicdiseases and, in particular, for the induction of a Th1 immune responseand the suppression or inhibition of a Th2/Th17 immune response.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto an active ingredient, a pharmaceutically acceptable excipient,carrier, buffer stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material will depend on the route ofadministration, which may be, for example, oral, intravenous, intranasalor via oral or nasal inhalation. The formulation may be a liquid, forexample, a physiologic salt solution containing non-phosphate buffer atpH 6.8-7.6, or a lyophilised or freeze-dried powder.

Dose

The composition is preferably administered to an individual in a“therapeutically effective amount” or a “desired amount”, this beingsufficient to show benefit to the individual. As defined herein, theterm an “effective amount” means an amount necessary to at least partlyobtain the desired response, or to delay the onset or inhibitprogression or halt altogether the onset or progression of a particularcondition being treated. The amount varies depending upon the health andphysical condition of the subject being treated, the taxonomic group ofthe subject being treated, the degree of protection desired, theformulation of the composition, the assessment of the medical situationand other relevant factors. It is expected that the amount will fall ina relatively broad range, which may be determined through routinetrials. Prescription of treatment, e.g. decisions on dosage etc., isultimately within the responsibility and at the discretion of generalpractitioners, physicians or other medical doctors, and typically takesaccount of the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners. The optimal dose can be determined byphysicians based on a number of parameters including, for example, age,sex, weight, severity of the condition being treated, the activeingredient being administered and the route of administration. A broadrange of doses may be applicable. Considering oral administration to ahuman patient, for example, from about 10 μg to about 1000 μg of agentmay be administered per human dose, optionally for 3 to 4 doses. Dosageregimes may be adjusted to provide the optimum therapeutic response andreduce side effects. For example, several divided doses may beadministered daily, weekly, monthly or other suitable time intervals orthe dose may be proportionally reduced as indicated by the exigencies ofthe situation.

Unless otherwise defined, all technical and scientific terms used hereinhave the meaning commonly understood by a person who is skilled in theart in the field of the present invention.

Autoimmune Disease

The term “autoimmune disease” as used herein is understood to mean anydisease or condition which is caused by a body's tissues being attackedby its own immune system.

Throughout the specification, unless the context demands otherwise, theterms “comprise” or “include”, or variations such as “comprises” or“comprising”, “includes” or “including” will be understood to imply theinclusion of a stated integer or group of integers, but not theexclusion of any other integer or group of integers.

The present invention will now be exemplified with reference to thefollowing non-limiting figures and examples which are provided for thepurpose of illustration and are not intended to be construed as beinglimiting on the present invention. Other embodiments of this inventionwill be apparent to those of ordinary skill in the art in view of thisdescription.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a graph of IgG subtype (IgG1 and IgG2a) production inBALB-c mice immunised with ovalbumin and different subtypes of IFN-α.

FIG. 2 shows a graph of the percentage of IgG subtype (IgG1 and IgG2a)produced in BALB-c mice immunised with ovalbumin and different subtypesof IFN-α.

FIG. 3 shows a graph of IgG2a production in BALB-c mice immunised withovalbumin and MULTIFERON™, glycosylated IFN-α14 and non-glycosylatedIFN-α14 administered via intraperitoneal injection.

FIG. 4 shows a graph of IgG1 production in BALB-c mice immunised withovalbumin and MULTIFERON™, glycosylated IFN-α14 and non-glycosylatedIFN-α14 administered via intraperitoneal injection.

FIG. 5 shows a graph of IgG2a production in BALB-c mice immunised withovalbumin and MULTIFERON™, glycosylated IFN-α14 and non-glycosylatedIFN-α14 administered orally.

FIG. 6 shows a graph of IgG1 production in BALB-c mice immunised withovalbumin and MULTIFERON™, glycosylated IFN-α14 and non-glycosylatedIFN-α14 administered orally.

FIG. 7 shows inhibition of human PBMC interleukin-17 (IL-17) secretionwith lipopolysaccharide (LPS) alone and with LPS and increasingconcentrations of IFN-a2a (black), IFN-α10 (white) or IFN-14 (grey).

FIG. 8 shows the inhibition of Interleukin-4 (IL4)-induced CD4+Th2 celldevelopment using increasing concentrations of IFN-α2a (black), IFN-α10(white) or IFN-14 (grey).

FIG. 9 shows the IFN-α10 and IFN-α14 hybrid amino acid sequence whichcontains the 2 interferon receptor (IFNaR1 and IFNaR2) binding sites.

Based on the protein sequence SEQ ID NO:1 using online webservices (forexample http://www.ebi.ac.uk/Tools/st/emboss_backtranseq/), a nucleicacid sequence can be obtained. FIG. 10 provides the reverse translationof the protein sequence SEQ ID NO:1 when an E. coli codon usage table isused.

FIG. 11 shows the alignment of a previously described IFNalpha10 andIFNalpha14 hybrid amino acid sequence with the IFNalpha10 and IFNalpha14hybrid amino acid sequence as presently claimed.

FIG. 12 indicates the effect caused by rIFN-α14 on the production ofIL-17 in whole human blood incubated with one microgram E. colilipopopolysaccharide (LPS) for 48 hours. The α-14 gave a significantsuppression of IL-17 secretion. IL-α-2 and α-10 showed no significantsuppression.

FIG. 13 shows the effect caused by rIFN-α14 on the production of IL-17from human peripheral blood mononuclear cells incubated with 10micrograms E. coli lipopolysaccharide (LPS) for 48 hours. The α-14caused a significant suppression of IL-17 secretion with and without LPSactivation. IL-α-2 and α-10 showed no significant changes in the IL-17concentrations (results not shown).

FIG. 14 shows the effect caused by rIFN-α10, rIFN-α14 and rIFN-α2 onIL-17 production by whole human blood incubated with PHA for 5 days. Theα-14 is an extremely potent inhibitor (P<0.001 at 1,000 IU/ml) of IL-17compared with the commonly available α-2; the α-10 is more than 20× lessactive in this context.

FIG. 15 shows the effect caused by rIFN-α10, rIFN-α14 and rIFN-α2 onIFN-gamma production by whole human blood incubated with PHA for 5 days.The α-10 is the most potent interferon—alpha in this context causingenhanced secretion of IFN-gamma—critical for both and innate andadaptive immunity against viruses, intracellular bacterial infectionsand in the control/elimination of tumours.

FIG. 16 illustrate a sequence alignment of IFN-alpha10 (SEQ ID NO:4) andIFN-alpha14 (SEQ ID NO:5) amino acid sequences and the hybrid sequenceSEQ ID NO: 1 discussed herein.

FIG. 17 shows the effect caused by rIFN-α14 and rIFN-α2 on IL-17production by whole human blood incubated with PHA for 5 days. The α-14is an extremely potent inhibitor of IL-17 compared with the commonlyavailable IFN-α2 (EC50s for the two subtypes are 100 and 10,000 IU/ml).

FIG. 18 shows the induction of Interferon-gamma by Interferon-alphasubtypes and in particular demonstrates the effect caused by IFN-α10,IFN-α14 and IFN-α2 on IFN-gamma production by whole human bloodincubated with PHA for 5 days. The α-10 is the most potentinterferon—alpha in this context causing enhanced secretion ofIFN-gamma.

FIG. 19 shows the effect of the IFN-α10-IFN-α14 hybrid of the presentinvention (SEQ ID NO:1) on IL-17 production from whole human bloodcompared to the effect on IL-17 of a previously disclosedIFN-α10-IFN-α14 hybrid. The hybrid of the present invention demonstratesan greater reduction in IL-17.

EXAMPLE 1—IDENTIFICATION OF INTERFERON-ALPHA SUBTYPES THAT AREIMMUNOLOGICAL ADJUVANTS

50 μg ovalbumin and 10⁵ IU of interferon subtypes IFN-α14, IFN-α2,IFN-α21, IFN-α10, an IFN “mix” (including IFN-α1, IFN-α8, IFN-α21 andpossibly IFN-α17), IFN-α8, Intron A, MULTIFERON™ and IFN-α1 in 50 μlwere administered via intraperitoneal injection three times per week toBALB-c female mice, in groups of 10.

The serum concentrations of IgG1 mg/ml (Th2 response—humoral immunity tothe ovalbumin antigen) and IgG2a mg/ml (Th1 response—cell-mediatedimmunity to the ovalbumin antigen) were measured by ELISA.

FIGS. 1 and 2 show the anti-ovalbumin IgG subtype production in BALB-cmice treated with IFN-α14, IFN-α2, INF-a21, IFN-α10, a “mix” of IFN-α1,IFN-α8, IFN-α21 and possibly IFN-α17), IFN-α8, Intron A, MULTIFERON™,ovalbumin only, ovalbumin plus human serum albumin (used as a carrier ininterferon preparations) and IFN-α1.

The inventor demonstrated that IFN-α10 and IFN-α14 enhanced theproduction of IgG2a antibodies significantly which is indicative of anenhanced Th1 immune response. The inventor also demonstrated thatIFN-α10 in particular showed low production of IgG1 antibody which isindicative of suppressing a Th2/Th17 immune response.

EXAMPLE 2—IDENTIFICATION OF ANTIBODY RESPONSE IN BALB-C MICE AFTERADMINISTRATION OF A COMPOSITION COMPRISING A FLU VACCINE AND A LOW DOSEOF LEUKOCYTE DERIVED INTERFERON-ALPHA (LDA1)

The standard flu vaccine was mixed with a low dose (10⁵ IU) of leukocytederived interferon alpha (LDA1). Without the interferon, a smallanti-flu antibody response was recorded in mice, approximately 50 timesless than with an injection. With interferon-alpha, the response fromthe orally delivered vaccine was exactly the same as the injectedvaccine. A series of buccal immunisations were carried out using astandard protein antigen (ovalbumin). Two interferons were compared,namely, the LDA1 and an isolated subtype, IFN-α14. Both produced aremarkable oral immunisation of the mice, but whereas the LDA1 gave abalanced response, the IFN-α14 gave only a significant humoral response.The production of IgG1 is indicative of a Th2/Th17 response (humoralimmunity) and the production of IgG2a is indicative of a Th1 response(cell-mediated immunity).

EXAMPLE 3—THE IDENTIFICATION OF IFN-ALPHA AS AN ORAL IMMUNOLOGICALADJUVANT

50 μg ovalbumin and 10⁵ IU of interferon subtypes, namely MULTIFERON™,glycosylated IFN-α14 and non-glycosylated IFN-α14, in 50 μl doses wereadministered three times a week to BALB-c female mice via oral (buccal)and intraperitoneal injection administration.

The controls used were antigen alone and Titermax—Titermax is a mixtureof compounds used in antibody generation and vaccination to stimulatethe immune system to recognise an antigen given together with themixture. Titermax is a recently developed immune adjuvant deemed to besafe in animals.

Serum concentrations (mg/ml) of IgG1 (indicative of a Th2/Th17 response)and IgG2a (indicative of a Th1 response) anti-ovalbumin antibodies werequantitated by ELISA.

The production of IgG2a and IgG1 antibodies when MULTIFERON™,glycosylated IFN-α14 and aglycosyl IFN-α14 (CHO cell-derived) wereadministered both orally and by injection were compared (see FIGS. 3, 4,5 and 6).

The inventor demonstrated that IFN-α14 showed pronounced immunologicaladjuvant activity both orally and by injection. No significantdifference was seen between the glycosylated and non-glycosylatedpreparations.

The inventor also demonstrated that IFN-α14 only enhanced IgG2aproduction associated with Th1 responses by the oral route ofadministration. Hence IFN-α14 is an activator of cell-mediated immunitywhen administered orally.

MULTIFERON™ enhanced both IgG1 and IgG2a responses when administeredboth orally and by injection i.e. it induced both Th1 and Th2 responsessignificantly.

EXAMPLE 4—IN VITRO DETERMINATION OF THE INHIBITION OF HUMORAL IMMUNITY(TH2/TH17) BY INTERFERON-ALPHA SUBTYPES—ANALYSIS OF TH17 LYMPHOCYTES ANDINTERLEUKIN 17

A total of 2×10⁶ human PBMCs were stimulated with lipopolysaccharide(LPS) in the absence or presence of increasing concentrations ofrecombinant human alpha-IFN. Supernatants were collected after 24 hoursand IL-17 concentrations measured by ELISA.

Human Cell Culture

Human peripheral blood was collected from healthy volunteers andperipheral blood mononuclear cells (PBMCs) were obtained by Lymphoprepgradient centrifugation (Pierce). For PBMC experiments, 2×10⁶ PBMCs perml were seeded in 24-well plates and stimulated with lipopolysaccharide(LPS) from Escherichia coli 055:B5 (Sigma) or 2×10⁶ PBMCs per mL wereseeded into 24-well plates and stimulated with 5 mg/mL plate-boundanti-CD3 (clone: UCHT1) and 2.5 mg/mL anti-CD28 (clone: CD28.2). Naive Tcells (CD4+CD45RA) were obtained by magnetically labeling and depletionof non-helper T-cell and memory T-cells performed according tomanufacturer's instructions (Miltenyi Biotec). A total of 1×10⁵ naiveT-cells were primed in 96-well flat bottom plates coated with anti-CD3(clone UCHT1, 2.5 mg/mL) and with anti-CD28 (clone CD28.2, 2.5 mg/mL)antibodies. After 48 h of culture, 20 IU/mL recombinant human IL-2(Peprotech) was added to the culture.

For human Th17 differentiation, cells were supplemented withneutralising anti-IL-4 and anti-IFNγ antibodies (both from Peprotech)and with 10 ng/mL recombinant IL-1β and 50 ng/mL recombinant IL-6 (bothfrom Peprotech). Where required, recombinant human IFNα 10/14 was addedto the culture. After 5 days of culture, cells were washed, transferredinto new plates and expanded until day 12 in the presence of 20 IU/mLrecombinant IL-2.

ELISA and Intracellular Cytokine Staining

The IL-17 producing capacity of primed Th17 cells was assessed bystimulation with 0.1 ng/ml LPS or alternatively can be assessed by thestimulation of human cells with soluble 1 mg/mL anti-CD3 (clone: OKT3)and phorbol-12-13-dibutyrate (PdBu). Concentrations of human IL-17 incell culture supernatants were determined using commercially availableantibody pairs and protein standards (R&D Systems). Absorption wasdetermined using an ELISA reader at 450 nm. For intracellular stainingof mouse IFNγ and IL-17, T-cells are stimulated with PMA and ionomycinfor 5 hours. Brefeldin A is added for the final 3 h of culture.Intracellular staining can be performed with a BD Cytofix/Cytoperm kitaccording to the manufacturer's instructions. Cells are incubated withfluorescein isothiocyanate-labeled anti-IFNγ (clone: XMG1.2, BDPharmingen) and Alexa Fluor 647-labeled anti-mouse IL-17A (clone:eBio17B7, eBioscience). After washing, cells are immediately analysedusing Fluorescence-activated cell sorting (FACS).

Results As shown by FIG. 7, inhibition of IL-17 was found to occur inthe order IFNα10>IFNα14>IFNα2a. P<0.05 (FIG. 7).

EXAMPLE 5—IN VITRO DETERMINATION OF THE INHIBITION OF HUMORAL IMMUNITY(TH2/TH17) BY INTERFERON-ALPHA SUBTYPES—ANALYSIS OF TH2 CELLS ANDASSOCIATED CYTOKINES CRTH2 Background

CRTH2 (Chemoattractant Receptor-homologous molecule expressed on Th2cells) is a G-protein coupled receptor expressed by Th2 lymphocytes,eosinophils, and basophils. The receptor mediates the activation andchemotaxis of these cell types in response to prostaglandin D2 (PGD2),the major prostanoid produced by mast cells. PGD2 is released throughmast cell degranulation in the initial phase of IgE-mediated reactions.This process is also thought to occur at the site of inflammation, suchas the nasal and bronchial mucosa. Through interaction with CRTH2, PGD2is thought to mediate recruitment and activation of CRTH2-bearing celltypes to the site of the allergic reaction, in consequence amplifyingand maintaining the allergic inflammation. In the nasal and bronchialmucosa, this pro-inflammatory cascade is thought to start during theso-called late allergic response occurring 3 to 9 hours after allergenchallenge. The interaction between PGD2 and CRTH2 would, therefore,contribute to the so-called “Th2 polarisation”, with consequent Th2cytokine production and the typical eosinophilic and basophiliccharacteristics of the inflammation.

IFNα Inhibits Human CD4+Th2 Development.

Purified human CD4+/CD4SRA+ cells were activated with plate-boundanti-CD3/anti-CD28 under defined cytokine conditions. Induction of CRTH2expression was assessed by flow cytometry. All P<0.05, above 100IU IFNcompared with IL-4 alone.

Human Subjects

Peripheral blood was collected from healthy adult donors and cellspurified as below.

T Cell Cultures and Analysis

Peripheral blood was obtained from healthy male adult donors and naiveCD4+/CD45RA+ T cells were purified (>92%) from buffy coats by magneticbead separation (BD Biosciences, USA). CD4+ cells were activated withplate-bound anti-CD3/anti-CD28 and IL-2 (50U/ml) in complete Iscove'sModified Dulbecco's Medium containing 10% FCS, in the presence ofrecombinant human recombinant IL-4 (R&D Systems, USA), at aconcentration of 20 ng/ml for 7 days. Flow cytometric analysis wasperformed with hCD294 (chemo-attractant receptor homologous moleculeexpressed on Th2 cells [CRTH2])-Alexa 647 (BD Biosciences).

Results

In humans, the PGD2 receptor, CRTH2, is selectively expressed on Th2cells and is induced by IL-4 during Th2 development. IL-4 promoted thedevelopment of cells expressing CRTH2. However, as shown in FIG. 8 allthe IFN-alphas markedly blocked IL-4 driven CRTH2 expression, in adose-dependent manner in the order IFNα10>IFNα14>IFNα2a, thus supportingthe concept that these cytokines suppress Th2 (humoral) immunity, butare recognised as potent activators of Th1-associated immunity.

As shown in FIG. 12, the effect of rIFN-α14 on the production of IL-17in human blood incubated with LPS for 48 h was tested.

Whole human blood was incubated without (open columns) or with 1 μg/mlLPS (cross hatched columns) in the absence and presence of a range ofconcentrations of rIFN-α14 (0-1,000 IU/ml) for 48 h at 37° C., in anatmosphere of 5% CO₂ in air, in a humidified incubator. Plasma wascollected by centrifugation and levels of IL-17 determined by ELISA.

FIG. 12 indicated a dose response to IFN-α14 wherein 1 mg=10⁻⁸IU.

As shown in FIG. 13 the effect of rIFN-α14 on the production of IL-17 inhuman PBMCs incubated with LPS for 48 h was tested.

Human Peripheral Blood Mononuclear cells (PBMCs), a critical componentin the immune system, were isolated from whole human blood by densitygradient centrifugation. 2×10⁶ PBMCs were incubated without (opencolumns) or with 10 μg/ml LPS (cross hatched columns) in the absence andpresence of a range of concentrations of rIFN-α14 (0-1,000 IU/ml) for 48h at 37° C., in an atmosphere of 5% CO₂ in air, in a humidifiedincubator. Levels of IL-17 in the supernatant were determined by ELISA.

As indicated by FIG. 13, increasing concentrations of rIFN-α14 was foundto reduce the IL-17 both in untreated and treated LPS cells.

As shown in FIG. 14, the effect of rIFNα10, rIFNα14 and rIFN2 on IL-17production by whole blood incubated with phytohaemagglutinin (PHA) for 5days was tested.

Whole human blood was diluted 1/10 with RPMI 1640 culture medium andincubated without or with 100 μg/ml PHA in the absence and presence of arange of concentrations of rIFN-α14, rIFN-α10 and rIFN-α2 for 5 days at37° C., in an atmosphere of 5% CO₂ in air, in a humidified incubator. Atthe end of this period, supernatants were aspirated and levels of IL-17in supernatants measured by ELISA. Values represent the mean±sem, forn=3 incubations. Statistical analysis and IC50 values were determinedusing GraphPad Prism 5 (GraphPad Software Inc., California, USA).

As indicated in FIG. 14 the provision of rIFN-α14 at higherconcentrations (100-1000IU/ml) caused a greater decrease in IL-17 thanthe provision of IFN-α2 or IFN-α10. rIFN-α14 is considered to be themost potent interferon tested at reducing IL-17 levels.

Similar results were achieved as shown in FIG. 17. Here the effect ofrIFNα14 and rIFN2 on IL-17 production by whole blood incubated withphytohaemagglutinin (PHA) for 5 days was tested using the samemethodology. As indicated in FIG. 17 the provision of rIFN-α14 at higherconcentrations (100-10001U/ml) caused a greater decrease in IL-17 thanthe provision of IFN-α2. rIFN-α14 is considered to be the most potentinterferon tested at reducing IL-17 levels.

As shown in FIG. 15, the effect of rIFNα10, rIFNα14 and rIFNα2 onIFN-gamma production by whole blood incubated with PHA for 5 days.

Whole human blood was diluted 1/10 with RPMI 1640 culture medium andincubated without or with 100 μg/ml PHA in the absence and presence of arange of concentrations of rIFN-α10, rIFN-α14 and rIFN-α2 for 5 days at37° C., in an atmosphere of 5% CO₂ in air, in a humidified incubator. Atthe end of this period supernatants were aspirated and levels ofIFN-gamma in supernatants measured by ELISA. Values represent themean±sem, for n=3 incubations. plasma was collected by centrifugationand levels of IFN-gamma determined by ELISA. Values represent themean±sem, for n=3 incubations.

It was determined that rIFN-α10 was the most effective of theinterferons tested at promoting levels of IFN-gamma. IFN-gamma haspreviously been suggested to be important in providing an anti-cancereffect.

Similar results were achieved as shown in FIG. 18. Here the effect ofrIFNα14 and rIFN2 on IFN-gamma production by whole blood incubated withphytohaemagglutinin (PHA) for 5 days was tested using the samemethodology. As indicated in FIG. 18 the provision of rIFN-α10 caused agreater increase in IFN-gamma than the provision of IFN-α2. rIFN-α10 isconsidered to be the most potent interferon tested at increasingIFN-gamma levels.

As shown in FIG. 19, the effect of the IFN-α10-IFN-α14 hybrid of thepresent invention (SEQ ID NO:1) on IL-17 production from whole humanblood was compared to the effect on IL-17 of the IFN-α10-IFN-α14 hybriddisclosed in PCT/GB2015/050717.

Whole human blood was diluted 1/10 with RPMI 1640 culture medium and wasincubated with PHA (100 μg/ml) in the presence of a range ofconcentrations of either the IFN-α10-IFN-α14 hybrid of the presentinvention (IFN alpha-hybrid 1) or the IFN-α10-IFN-α14 hybrid disclosedin PCT/GB2015/050717 (IFN alpha-hybrid 2) for 5 days at 37° C. in anatmosphere of 5% CO₂ in air in a humidified incubator. At the end ofthis period supernatants were collected and levels of IL-17 measured byELISA. Values represent mean±sem, for n=3 incubations. P<0.05 for allpoints of the data between the two hybrids except 100 IU/ml, which wasnon significant. The Red diamonds indicate IFN-α10-IFN-α14 hybrid 1 andthe blue circles indicate IFN-α10-IFN-α14 hybrid 2.

It was determined that the IFN-α10-IFN-α14 hybrid of the presentinvention demonstrates a greater reduction in IL-17.

EXAMPLE 6—EFFECTS OF HUMAN INTERFERON ALPHA-14 AND ALPHA-10 ONUNSTIMULATED AND ACTIVATED HUMAN MONONUCLEAR LEUKOCYTES FROM NORMALSUBJECTS

TABLE 1 Synopsis of 400 interleukins, chemokines and protein markerestimations* following IFN-α10/14 treatment of human mononuclear cellsFOLD NUMBER OF FOLD NUMBER PHA-STIMULATED/ OF UNSTIMULATED/PHA-STIMULATED ALPHA-IFN ALPHA-IFN ANALYTE TREATED CELLS TREATED CELLSCYTOKINES Alpha-14 Alpha-10 Alpha-14 Alpha-10 IL-1a 0 +23 1 +2 IL-1b 0+70 −2 1 IL-1(F5 to F10) 0 0 0 0 IL-2 0 0 +7 +4 IL-3 0 0 −11 x IL-4 0 01 −3 IL-5 0 0 −420 1 IL-6 −19 +1000 1 1 IL-7 0 0 0 0 IL-8 1 +100 1 1IL-9 0 0 0 0 IL-10 0 +5 +2 +2 IL-11 0 0 0 0 IL-12 p40 0 +350 0 +1 IL-12p70 0 0 +11 0 IL-13 0 0 −5 1 IL-15 0 0 0 0 IL-16 1 1 1 1 IL-17 0 0 −43−5 IL-18 0 0 0 0 IL-20 0 0 0 0 IL-21 0 x 0 x IL-23 +4 +3 +6 1 IL-24 0 00 0 IL-27 0 1 0 1 IL-28 0 0 0 0 IL-29 0 0 0 0 IL-31 0 0 0 0 IL-33 0 0 00 IL-34 0 0 0 0 IFN-gamma 0 0 +600 +3000 G-CSF −1500 +20 1 1 GM-CSF 0 00 1 CD MARKERS CD14 +2 +2 +2 +2 CD23 −22 1 −850 −3 CD30 0 0 0 0 CD40 +2+2 1 1 CD97 −2 1 −5 −5 CD152 (CTLA-4) 1 0 −2 0 CD154 1 x +2 x CD163 −2 1−2 1 CD200 1 1 −1 1 CD223 (LAG3) 0 0 −3 +3 SELECTED CHEMOKINES ANDPROTEINS CXCL1 (GROa) −7600 −12 −3400 1 CXCL5 (ENA-78) −6 +16 −32 −3CXCL10 (IP10) +460 +10 +1 1 CCL1 (I-309) 0 1 −24 1 CCL7 (MCP-3) −2 −200−149 1 CCL16 (HCC-4) 0 1 −100 1 CCL20 (MIP-3a) −69 +40 −2 1 MMP-2 +600+200 +450 +500 (collagenase) MMP-10 −121 1 −2 −2 (proteoglycanase) ACE-2+12 0 +6 0 PDGF Ralpha −4 −7 −1 −3 Tie-1 −170 −200 −8 −280 ICAM-1 −1 +2−3 1 TREM-1 +5 −2 +2 −5 E-SELECTIN 1 −7 1 −2 0 = no analyte detected 1 =analyte present but no effect of alpha-10/14 x = not determined Thepositive effect of alpha-10/14 is denoted by a + The negative effect ofalpha-10/14 interferon is denoted by a − *The assay system used was theRayBio Quantibody Human Cytokine Array 9000 (QAH-CAA-9000 provided byInsight Bio Ltd.). This a multiplex ELISA, measuring the concentrationsof 400 proteins in a single assay process, including pro- andanti-inflammatory markers, interleukins, cancer markers, chemokines,growth factors and related molecules. Human peripheral blood mononuclearleukocytes (normal blood donors) were treated with 10 ng/ml IFN-α14/10for 4 hours prior to assay. Tests were performed on 2 groups of cells -a) unactivated and b) activated with PHA (phytohaemagglutinin) to inducea high level of stimulation.

Effects of Alpha-14 on Activated Immune Cells

More than 30 interleukins were quantitated but only 6 showed significantchanges in the activated cells, indicating the targeted and veryspecific nature of the interaction of the alpha-14 with the human immuneresponse.

Interleukin 2 increased by 7-fold, IL-12p70 +11 fold and interferon-γ+600 fold, indicating a strong proliferation of the Th1 (cell-mediated)response while a 6-fold increase in IL-23 is in keeping with its role incell-mediated immunity and its association with IL-12.

Very large decreases were observed with IL-3 and IL-5 of 11 and 420-foldrespectively. These molecules are associated with the production ofmyeloid cells and immunoglobulin production (humoral immunity). IL13also decreased by 5-fold, which is important as this interleukin isimplicated in the secretion of IgE, the allergy antibody. Also crucialwas the 43-fold decrease in IL-17. This regulatory cytokine is increasedin autoimmune diseases, humoral (antibody-mediated) immunity andstimulation of inflammation through attraction of neutrophils.

CD23 or FcεRII is a receptor for the allergy antibody, IgE, and isdisplayed widely on different types of leukocytes. CD23 activationcontrols IgE production and significant increases are seen in patientswith allergic disorders. This important marker was decreased by850-fold, in the activated cells, by alpha-14.

Effects of IFN Alpha-14 on Non-Activated Immune Cells

IL-6 decreased 19-fold. This cytokine stimulates liver protein synthesisin responses to traumas, causes increases in body temperature and isinvolved in muscle contraction. However, it is its essential role inantibody-mediated immunity that is important in allergy.

G-CSF was also decreased by more than 1000-fold. This molecule canstimulate the bone marrow to make increased numbers of neutrophils thatcould be involved in inflammation. At the same time the secretion of thechemokine CXCL1 was suppressed by 7,500-fold—this prevents it attractingneutrophils to the site of a response and causing inflammation. Also theconcentration of the chemokine, CXCL10 was enhanced by 460-fold—its roleis to attract T-lymphocytes to an ongoing immune response.

Effects of IFN Alpha-10 on Activated Immune Cells

As with alpha-14, alpha-10 only regulated a small number of cytokinesout of the numbers assessed. Of particular note were the increases inIL-2 and interferon-γ of 4 and 3000 fold respectively indicating aswitch to cell-mediated immunity. IL-17 levels fell by 5 fold,confirming this change in balance.

The large reduction in CD23 was not evident with alpha-10 and its majoreffects on chemokines were on Tie-1 (tyrosine kinase crucial in theprocess of lymphatic remodelling) and TREM-1 (neutrophil activation)where it caused reductions of 280 and 5 fold respectively.

Effects of Alpha-10 on Non-Activated Immune Cells

Alpha-10 showed significant activity in this context enhancing IL-1α/βby up to 70 fold and IL-6,8,10,12 (p40) by 1000, 100, 5 and 350 fold inkeeping with a strong support for cell-mediated over humoral immunity.G-CSF was also enhanced by 20 fold in total contrast to alpha-14.

Few changes were recorded with the CD markers but CXCL1 was reduced by12 fold while CXCL5 and 10 increased by 16 and 10 fold and CCL20 rose by40 fold. However, CCL7 and Tie-1 fell by 200 fold each. These resultsare in keeping with a significant movement towards cell-mediatedimmunity.

Result

The low doses of interferon-alpha 14 and 10 have modified cytokinesynthesis in order to enhance cell-mediated immunity at the expense ofantibody-mediated immunity. This would be invaluable in enhancing theactivities of certain vaccines where a humoral immune response can bedetrimental e.g. viral and cancer vaccines.

In addition the results are totally in keeping with the generalunderstanding that allergy can be alleviated by changing the immuneresponse to an allergen by shifting an antibody response to a cellularresponse. Such a change would be part of acquired immunity and hence,potentially, a long-term solution by developing tolerance.

In addition, the alpha-14 significantly suppressed the capacity ofleukocytes to make/utilise IgE and hence it inhibited the immediateeffects of an allergic reaction, together with reducing inflammatoryelements of immunity while enhancing the involvement of more controlelements.

All documents referred to in this specification are herein incorporatedby reference.

Various modifications and variations to the described embodiments of theinventions will be apparent to those skilled in the art withoutdeparting from the scope of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes of carrying out the invention which are obvious tothose skilled in the art are intended to be covered by the presentinvention.

1. A method for the treatment and/or prophylaxis of a condition where anenhancement of a Th1-mediated immune response and suppression of aTh2/Th17-mediated immune response are desired, said method comprisingthe step of: (i) administering to a subject in need thereof atherapeutically effective amount of at least one hybrid interferon alpha(IFN-α), wherein the hybrid IFN-α contains a IFN-α10 receptor bindingsite and a IFN-α14 receptor binding site, and wherein the hybrid IFN-αhas increased affinity for interferon receptor 1 (IFNR1) and interferonreceptor 2 (IFNR-1) compared to IFN-α10 and IFN-α14.
 2. The method ofclaim 1, wherein the condition where an enhancement of a Th1-mediatedimmune response and suppression of a Th2/Th17-mediated immune responseare desired is selected from the group consisting of: an autoimmunedisease, an inflammatory disease, bowel disease, ulcerative colitis,Crohn's disease, an allergy, a food allergy, an allergic disease,cancer, hepatic cell cancer, lung cancer, non-small cell lung cancer,ovarian cancer, breast cancer, skin cancer, melanoma, genitourinarycancer, prostate cancer, renal cell cancer, or bladder cancer. 3-6.(canceled)
 7. The method of claim 1, wherein the method furthercomprises administering to the subject a therapeutically effectiveamount of a vaccine composition for treatment or prophylaxis of thecondition where an enhancement of a Th1-mediated immune response andsuppression of a Th2/Th17-mediated immune response are desired.
 8. Themethod of claim 7, wherein the vaccine composition comprises at leastone allergen or antigen capable of mediating a Th2/Th17 immune response,a food allergen, a tumour antigen, a tumour specific antigen, or atumour-associated antigen.
 9. (canceled)
 10. (canceled)
 11. The methodof claim 1, wherein the hybrid IFN-α is administered orally. 12-15.(canceled)
 16. The method of claim 1, wherein the hybrid IFN-α comprisesa IFN-α10-IFN-α14 hybrid, wherein the IFN-α10-IFN-α14 hybrid comprisesthe amino acid sequence of SEQ ID NO:3 having one or more of thefollowing amino acid substitutions: a Glu to Lys mutation at position 71of SEQ ID NO:3, a Glu to Asp mutation at position 78 of SEQ ID NO:3, aGln to Glu mutation at position 79 of SEQ ID NO:3, a Ser to Tyr mutationat position 86 of SEQ ID NO:3, and a Arg to Lys mutation at position 121of SEQ ID NO:3.
 17. (canceled)
 18. (canceled)
 19. A hybrid IFN-αcomprising a IFN-α10 receptor binding site and a IFN-α14 receptorbinding site, wherein the IFN-α has increased affinity for interferonreceptor 1 (IFNR1) and interferon receptor 2 (IFNR-1) compared toIFN-α10 and IFN-α14, and wherein the hybrid IFN-α enhances one or moreTh1-mediated immune responses and suppresses one or moreTh2/Th17-mediated immune responses. 20-24. (canceled)
 25. Thepharmaceutical composition of claim 91, wherein the hybrid IFN-α isformulated for use in combination with a vaccine composition.
 26. Thepharmaceutical composition of claim 25, wherein the vaccine compositioncomprises at least one allergen or antigen capable of mediating aTh2/Th17 immune response there against.
 27. The pharmaceuticalcomposition of claim 26, wherein the at least one allergen or antigencomprises a food allergen, a tumour antigen, tumour-specific antigen, ora tumour-associated antigen.
 28. The pharmaceutical composition of claim91, wherein the hybrid IFN-α is formulated for oral administration. 29.(canceled)
 30. The hybrid IFN-α of claim 19, wherein the hybrid IFN-αcomprises the amino acid sequence of SEQ ID NO: 1 or a fragment orvariant thereof.
 31. (canceled)
 32. (canceled)
 33. The hybrid IFN-α ofclaim 19, wherein the IFN-α comprises a IFN-α10-IFN-α14 hybrid, whereinthe IFN-α10-IFN-α14 hybrid comprises the amino acid sequence of SEQ IDNO:3 having one or more of the following amino acid substitutions: a Gluto Lys mutation at position 71 of SEQ ID NO:3, a Glu to Asp mutation atposition 78 of SEQ ID NO:3, a Gln to Glu mutation at position 79 of SEQID NO:3, a Ser to Tyr mutation at position 86 of SEQ ID NO:3, and an Argto Lys mutation at position 121 of SEQ ID NO:3.
 34. A method ofenhancing a Th1-mediated immune response and suppressing aTh2/Th17-mediated immune response in a subject comprising administeringto the subject the hybrid IFN-α of claim
 19. 35. The method of claim 34,wherein the subject has an autoimmune disease, an inflammatory disease,inflammatory bowel disease, ulcerative colitis, Crohn's disease, anallergy, a food allergy, an allergic disease, cancer, hepatic cellcancer, lung cancer, non-small cell lung cancer, ovarian cancer, breastcancer, skin cancer, melanoma, genitourinary cancer, prostate cancer,renal cell cancer, or bladder cancer. 36-39. (canceled)
 40. The methodof claim 34, wherein the method further comprises administering avaccine to the subject, wherein the vaccine comprises at least oneallergen or antigen capable of mediating a Th2/Th17 immune responsethere against. 41-61. (canceled)
 62. The pharmaceutical composition ofclaim 91, wherein the pharmaceutical composition further comprises apharmaceutically acceptable excipient, diluent, or carrier. 63.(canceled)
 64. A method for the treatment and/or prophylaxis of acondition mediated by enhanced expression of IL-17, said methodcomprising the step of: (i) administering to a subject in need thereof atherapeutically effective amount of at least one hybrid IFN-α, whereinthe hybrid IFN-α contains a IFN-α10 receptor binding site and a IFN-α14receptor binding site, and wherein the hybrid IFN-α has increasedaffinity for IFNR1 and IFNR-1 compared to IFN-α10 and IFN-α14.
 65. Themethod as claimed in claim 64 wherein the condition mediated by enhancedexpression of IL-17 is inflammatory bowel disease, ulcerative colitis,Crohn's disease, cancer, hepatic cell cancer, lung cancer, non-smallcell lung cancer, ovarian cancer, breast cancer, skin cancer, melanoma,genitourinary cancer, prostate cancer, renal cell cancer, or bladdercancer. 66-90. (canceled)
 91. A pharmaceutical composition comprisingthe hybrid IFN-α of claim 19.